A variety of hereditary defects in enzymes of pyruvate metabolism have been described which are associated with lactic acidosis and abnormal development. In this project we propose to apply basic biochemical information concerning the enzymes of pyruvate metabolism to detect and further characterize the nature of the defects involved and to correlate these defects with their physiological and clinical effects. Our effort will be concentrated on pyruvate dehydrogenase complex and pyruvate carboxylase, the two major enzymes of pyruvate metabolism. We will attempt to characterize defects of pyruvate dehydrogenase complex according to the activity and content of each of the three components of the complex, measuring their content with specific antibodies. Family studies will be carried out to measure enzyme levels in fibroblasts or leukocytes. In vitro assays in cells and tissue biopsies will be correlated with in vivo estimates of metabolic flux through the pathways involved. This will be done by monitoring the transition from pyruvate to fatty acid oxidation which occurs during fasting, using respiratory calorimetry and measuring metabolic flux with stable isotopes. Metabolism of labeled (13)C-Alpha-aminobutyrate will be measured since utilization of this analogue of pyruvate appears to depend on pyruvate dehydrogenase complex activity. Once characterized biochemically the turnover rates of these enzymes and levels of poly (A)+ RNA will be determined in fibroblasts from patients with defects. To explore the nature of defects of pyruvate dehydrogenase at the gene level, we plan to construct, isolate, and characterize cDNA clones specific for the two non-identical subunits of pyruvate dehydrogenase. This protocol will require partial amino acid sequence data of these two subunits in order to design mixtures of synthetic deoxyoligonucleotides which will be complementary to pyruvate dehydrogenase mRNA. We will use these cDNA probes to measure the level of PDH mRNA and gene structure in deficient individuals. These studies will lead to a more preciso knowledge of the basic causes and consequence of defects of pyruvate metabolism, which will be relevant to improved approaches to diagnosis and possible intervention in these disorders.